DNA polymerase I, the product of the PolA gene, is an important enzyme involved in both DNA repair and semiconservative DNA replication, in which the enzyme provides gap filling on the lagging strand. Besides its considerable biological interest, DNA polymerase I has engendered much research in terms of commercial applications. For example, the enzyme from E. coli, in the form of the Klenow fragment, is an essential reagent in molecular cloning since it is necessary for tasks such as the end filling of restriction fragments and other forms of gap filling. In addition, thermostabile DNA polymerase I genes are necessary for techniques and procedures involving the polymerase chain reaction (PCR).
Syphilis is an infectious venereal disease caused by the spirochete, Treponema pallidum. Syphilis is usually transmitted by sexual intercourse or acquired congenitally. If left untreated, the disease can ultimately lead to the degeneration of bones, heart, nerve tissue and other organs or tissues.
Little is known about the molecular biology of Treponema pallidum and less about the mechanisms of DNA synthesis and repair in this spirochete. The T. pallidum organism has an extremely long generation time (generally estimated at around 30 hours), but the limitations on its growth rate are unknown (1). It was hypothesized some 20 years ago from fragmentary data on the rate of DNA synthesis in a suboptimal in vitro culture system, that DNA synthesis is very slow in T. pallidum (2). DNA repair in T. pallidum appears to be defective with regard to oxidative lesions (3), but little else is known. This is unfortunate, since defects in DNA repair may relate to the fact that this treponeme cannot be grown in a cell free system, or be maintained at present even in the presence of tissue culture cells (4). Therefore, the isolation of the gene for DNA polymerase I from T. pallidum could be very important in answering questions about DNA replication and repair since it is important in both of these essential functions.
The DNA polymerase I enzyme, in general, is known to be involved in several important pathways of DNA repair and gap filling on the lagging strand during DNA replication (5). The gene has been found in all bacteria examined with the exception of a few species of Mycoplasmas and Archaebacteria (6,7). In most organisms, the enzyme contains three distinct domains: a 5'-3' exonuclease (important in removing damaged strands of DNA repair and removing RNA primers in replication); a 3'-5' exonuclease (which proofreads the DNA resulting from polymerization by the enzyme itself); and the polymerase domain, organized in this order from amino to carboxyl terminus of the protein (5). Because of the three domain structure, these enzymes are very large. The proofreading domain appears to be missing from the DNA polymerase I enzymes from Thermus aquaticus, Mycobacterium tuberculosis, and Mycobacterium leprae (8,9,10). The polymerase domain is highly conserved in all of the sequenced genes. There is more variability in the other domains, but specific amino acid motifs are found (11).
Due to the lack of a feasible system for growing T. pallidum in a clinical setting (T. pallidum can only be grown in tissue culture and cannot be serially passaged (4)), clinical diagnosis has traditionally depended on serological testing for antibody against T. pallidum (13). Direct testing for the presence of T. pallidum has been largely limited to darkfield examination of the primary chancre for the presence of spirochetes having the morphology of T. pallidum (13). This test lacks sensitivity and requires personnel training and experience to achieve accurate results. Consequently, this has led to the search for a PCR based test which should be highly sensitive and, with the choice of the proper target, could be very specific.
Orle et al. have reported the use of a PCR based test for the detection of syphilis (14). The specificity of this test is dependent on the choice of primers. In addition, U.S. Pat. Nos. 4,868,118; 5,350,842; and 5,508,168 describe a PCR based technique for clinical detection of T. pallidum. All of these patents involve the use of the 47 kD major immunogen, which is believed to be a carboxypeptidase involved in cell wall synthesis, and is further a penicillin binding protein (PBP) (19). Although this protein shows some cross-reactivity both immunologically and by PCR, it has no clear homologues by DNA sequence. This can be a major difficulty in cases of cross-reactivity since primers can only be selected for PCR by trial and error, i.e. known conserved and variant sequences are not known. This problem is inherent in all of the PCR based tests for T. pallidum described above. In no case does the gene used have clear homologues among known proteins. This is due to the fact that all of these genes were cloned after being identified as targets for an antibody response from the human host, none were cloned on the basis of function.
Therefore, there is a need for sensitive, specific methods for the detection of T. pallidum. Such methods would be particularly useful for facilitating a clinical diagnosis of syphilis. In addition, there is a need for probes and primers specific for T. pallidum to be used in detection methods and as scientific research tools to investigate the T. pallidum organism and to develop therapies and treatments.